Alternative splicing (AS) and processing of pre-messenger RNAs explains the discrepancy between the number of genes and proteome complexity in multicellular eukaryotic organisms. However, relatively few alternative protein isoforms have been experimentally identified, particularly at the protein level. In this study, we assess the ability of proteomics to inform on differently spliced protein isoforms in human and four other model eukaryotes. The number of Ensembl-annotated genes for which proteomic data exists that informs on AS exceeds 33% of the alternately spliced genes in the human and worm genomes. Examining AS in chicken via proteomics for the first time, we find support for over 600 AS genes. However, although peptide identifications support only a small fraction of alternative protein isoforms that are annotated in Ensembl, many more variants are amenable to proteomic identification. There remains a sizeable gap between these existing identifications (10–52% of AS genes) and those that are theoretically feasible (90–99%). We also compare annotations between Swiss-Prot and Ensembl, recommending use of both to maximize coverage of AS. We propose that targeted proteomic experiments using selected reactions and standards are essential to uncover further alternative isoforms and discuss the issues surrounding these strategies.